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- PDB-7rtn: Cryo-EM structure of bluetongue virus capsid protein VP5 at low e... -

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Basic information

Entry
Database: PDB / ID: 7rtn
TitleCryo-EM structure of bluetongue virus capsid protein VP5 at low endosomal pH
ComponentsOuter capsid protein VP5
KeywordsVIRAL PROTEIN / Bluetongue virus / capsid / membrane penetration / VP5
Function / homologyOuter capsid protein VP5, Orbivirus / Orbivirus outer capsid protein VP5 / viral capsid / structural molecule activity / Outer capsid protein VP5
Function and homology information
Biological speciesBluetongue virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsXia, X. / Wu, W.N. / Cui, Y.X. / Roy, P. / Zhou, Z.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386/DE028583/DE025567 United States
CitationJournal: Nat Microbiol / Year: 2021
Title: Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry.
Authors: Xian Xia / Weining Wu / Yanxiang Cui / Polly Roy / Z Hong Zhou /
Abstract: Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration ...Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4-3.9 Å show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated β-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses.
History
DepositionAug 13, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 3, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Assembly

Deposited unit
A: Outer capsid protein VP5
B: Outer capsid protein VP5
C: Outer capsid protein VP5


Theoretical massNumber of molelcules
Total (without water)177,2113
Polymers177,2113
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Outer capsid protein VP5


Mass: 59070.371 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Bluetongue virus (serotype 1 / isolate South Africa)
References: UniProt: K7QP12

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Bluetongue virus (serotype 1 / isolate South Africa) / Type: VIRUS
Details: The virus was generated from BHK-21 cell and purified by sucrose gradient.
Entity ID: all / Source: NATURAL
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Bluetongue virus (serotype 1 / isolate South Africa)
Strain: BTV-1
Details of virusEmpty: YES / Enveloped: NO / Isolate: SPECIES / Type: VIRION
Natural hostOrganism: sheep
Virus shellDiameter: 880 nm
Buffer solutionpH: 5.5
Buffer componentConc.: 20 mM / Name: sodium citrate
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3609
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
11RELION3.1classification
12RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 913440
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 572331 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model buildingPDB-ID: 3J9E

3j9e


Accession code: 3J9E / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036144
ELECTRON MICROSCOPYf_angle_d0.6198343
ELECTRON MICROSCOPYf_dihedral_angle_d4.563870
ELECTRON MICROSCOPYf_chiral_restr0.044930
ELECTRON MICROSCOPYf_plane_restr0.0041107

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